Nucleic acid molecules encoding a wheat sucrose transporter

ABSTRACT

This invention relates to an isolated nucleic acid fragment encoding a sucrose transport protein. The invention also relates to the construction of a chimeric gene encoding all or a portion of the sucrose transport protein, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the sucrose transport protein in a transformed host cell.

This application claims the benefit of U.S. Provisional Application No.60/081,148, filed Apr. 9, 1998.

FIELD OF THE INVENTION

This invention is in the field of plant molecular biology. Morespecifically, this invention pertains to nucleic acid fragments encodingsucrose transport proteins in plants and seeds.

BACKGROUND OF THE INVENTION

Sucrose is the first form of carbohydrate to leave photosynthesizingcells in most higher plants and is the main form of transported carbonin most annual field crops plants such as corn, soybeans and wheat. Assuch its movement and concentration across various plant membranes iscritical to plant growth and development. In addition sucrose is themain form of carbon that moves into developing seeds of soybeans, cornand wheat. This movement and concentration is accomplished by the actionof sucrose carrier proteins that act to move sucrose against aconcentration gradient by coupling sucrose movement to the oppositevectoral movement of a proton. Specific sucrose carrier sequences fromthese crop plants should find use in controlling the timing and extentof phenomena such as grain fill duration that are important factors incrop yield and quality. Accordingly, the availability of nucleic acidsequences encoding all or a portion of these enzymes would facilitatestudies to better understand carbohydrate metabolism and function inplants, provide genetic tools for the manipulation of these biosyntheticpathways, and provide a means to control carbohydrate transport anddistribution in plant cells.

SUMMARY OF THE INVENTION

The instant invention relates to isolated nucleic acid fragmentsencoding proteins involved in sucrose transport. Specifically, thisinvention concerns an isolated nucleic acid fragment encoding a sucrosetransport protein. In addition, this invention relates to a nucleic acidfragment that is complementary to the nucleic acid fragment encoding thesucrose transport protein. An additional embodiment of the instantinvention pertains to a polypeptide encoding all or a substantialportion of a sucrose transport protein.

In another embodiment, the instant invention relates to a chimeric geneencoding a sucrose transport protein, or to a chimeric gene thatcomprises a nucleic acid fragment that is complementary to a nucleicacid fragment encoding a sucrose transport protein, operably linked tosuitable regulatory sequences, wherein expression of the chimeric generesults in production of levels of the encoded protein in a transformedhost cell that is altered (i.e., increased or decreased) from the levelproduced in an untransformed host cell.

In a further embodiment, the instant invention concerns a transformedhost cell comprising in its genome a chimeric gene encoding a sucrosetransport protein, operably linked to suitable regulatory sequences.Expression of the chimeric gene results in production of altered levelsof the encoded protein in the transformed host cell. The transformedhost cell can be of eukaryotic or prokaryotic origin, and include cellsderived from higher plants and microorganisms. The invention alsoincludes transformed plants that arise from transformed host cells ofhigher plants, and seeds derived from such transformed plants.

An additional embodiment of the instant invention concerns a method ofaltering the level of expression of a sucrose transport protein in atransformed host cell comprising: a) transforming a host cell with achimeric gene comprising a nucleic acid fragment encoding a sucrosetransport protein; and b) growing the transformed host cell underconditions that are suitable for expression of the chimeric gene whereinexpression of the chimeric gene results in production of altered levelsof sucrose transport protein in the transformed host cell.

An addition embodiment of the instant invention concerns a method forobtaining a nucleic acid fragment encoding all or a substantial portionof an amino acid sequence encoding a sucrose transport protein.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE DESCRIPTIONS

The invention can be more fully understood from the following detaileddescription and the accompanying drawings and Sequence Listing whichform a part of this application.

FIG. 1A-FIG. 1F shows a comparison of the amino acid sequences set forthin SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 and theDaucus carota (SEQ ID NO:25), Oryza sativa (SEQ ID NO:26), Ricinuscommunis (SEQ ID NO:27) and Vicia faba (SEQ ID NO:28) sucrose transportprotein amino acid sequences.

The following sequence descriptions and sequence listings attachedhereto comply with the rules governing nucleotide and/or amino acidsequence disclosures in patent applications as set forth in 37 C.F.R.§1.821-1.825.

SEQ ID NO:1 is the nucleotide sequence comprising the entire cDNA insertin clone cepe7.pk0015.d10 encoding an entire corn sucrose transportprotein.

SEQ ID NO:2 is the deduced amino acid sequence of a sucrose transportprotein derived from the nucleotide sequence of SEQ ID NO:1.

SEQ ID NO:3 is the nucleotide sequence comprising a portion of the cDNAinsert in clone cr1n.pk0075.f5 encoding a portion of a corn sucrosetransport protein.

SEQ ID NO:4 is the deduced amino acid sequence of a portion of a sucrosetransport protein derived from the nucleotide sequence of SEQ ID NO:3.

SEQ ID NO:5 is the nucleotide sequence comprising a portion of the cDNAinsert in clone cr1n.pk0095.c10 encoding a portion of a corn sucrosetransport protein.

SEQ ID NO:6 is the deduced amino acid sequence of a portion of a sucrosetransport protein derived from the nucleotide sequence of SEQ ID NO:5.

SEQ ID NO:7 is the nucleotide sequence comprising the entire cDNA insertin clone r1r2.pk0043.b1 encoding a portion of a rice sucrose transportprotein.

SEQ ID NO:8 is the deduced amino acid sequence of a portion of a sucrosetransport protein derived from the nucleotide sequence of SEQ ID NO:7.

SEQ ID NO:9 is the nucleotide sequence comprising the entire cDNA insertin clone r1s6.pk0076.e2 encoding an entire rice sucrose transportprotein.

SEQ ID NO:10 is the deduced amino acid sequence of a sucrose transportprotein derived from the nucleotide sequence of SEQ ID NO:9.

SEQ ID NO: 11 is the nucleotide sequence comprising the entire cDNAinsert in clone sfl1.pk0001.g1 encoding an entire soybean sucrosetransport protein.

SEQ ID NO:12 is the deduced amino acid sequence of a sucrose transportprotein derived from the nucleotide sequence of SEQ ID NO:11.

SEQ ID NO:13 is the nucleotide sequence comprising a contig assembledfrom the cDNA inserts in clones sfl1.pk0043.c7 and sdp3c.pk012.c13encoding a portion of a soybean sucrose transport protein.

SEQ ID NO:14 is the deduced amino acid sequence of a portion of asucrose transport protein derived from the nucleotide sequence of SEQ IDNO:13.

SEQ ID NO:15 is the nucleotide sequence comprising a portion of the cDNAinsert in clone vs1n.pk0002.h3 encoding a portion of a Vernonia sucrosetransport protein.

SEQ ID NO:16 is the deduced amino acid sequence of a portion of asucrose transport protein derived from the nucleotide sequence of SEQ IDNO:15.

SEQ ID NO:17 is the nucleotide sequence comprising the entire cDNAinsert in clone w1e1n.pk0007.h8 encoding a portion of a wheat sucrosetransport protein.

SEQ ID NO:18 is the deduced amino acid sequence of a portion of asucrose transport protein derived from the nucleotide sequence of SEQ IDNO:17.

SEQ ID NO:19 is the nucleotide sequence comprising the entire cDNAinsert in clone w1e1n.pk0103.c11 encoding an entire wheat sucrosetransport protein.

SEQ ID NO:20 is the deduced amino acid sequence of a sucrose transportprotein derived from the nucleotide sequence of SEQ ID NO:19.

SEQ ID NO:21 is the nucleotide sequence comprising the entire cDNAinsert in clone w1m24.pk0015.g11 encoding an entire wheat sucrosetransport protein.

SEQ ID NO:22 is the deduced amino acid sequence of a sucrose transportprotein derived from the nucleotide sequence of SEQ ID NO:21.

SEQ ID NO:23 is the nucleotide sequence comprising the entire cDNAinsert in clone w1mk1.pk0002.e11 encoding an entire wheat sucrosetransport protein.

SEQ ID NO:24 is the deduced amino acid sequence of a sucrose transportprotein derived from the nucleotide sequence of SEQ ID NO:23.

SEQ ID NO:25 is the amino acid sequence of a Daucus carota sucrosetransport protein (NCBI Identifier No. gi 2969887).

SEQ ID NO:26 is the amino acid sequence of a Oryza sativa sucrosetransport protein (NCBI Identifier No. gi 2723471).

SEQ ID NO:27 is the amino acid sequence of a Ricinus communis sucrosetransport protein (NCBI Identifier No. gi 542020).

SEQ ID NO:28 is the amino acid sequence of a Vicia faba sucrosetransport protein (NCBI Identifier No. gi 1935019).

The Sequence Listing contains the one letter code for nucleotidesequence characters and the three letter codes for amino acids asdefined in conformity with the IUPAC-IUBMB standards described inNucleic Acids Research 13:3021-3030 (1985) and in the BiochemicalJournal 219 (No. 2):345-373 (1984) which are herein incorporated byreference. The symbols and format used for nucleotide and amino acidsequence data comply with the rules set forth in 37 C.F.R. §1.822.

DETAILED DESCRIPTION OF THE INVENTION

In the context of this disclosure, a number of terms shall be utilized.As used herein, an “isolated nucleic acid fragment” is a polymer of RNAor DNA that is single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases. An isolated nucleicacid fragment in the form of a polymer of DNA may be comprised of one ormore segments of cDNA, genomic DNA or synthetic DNA. As used herein,“contig” refers to an assemblage of overlapping nucleic acid sequencesto form one contiguous nucleotide sequence. For example, several DNAsequences can be compared and aligned to identify common or overlappingregions. The individual sequences can then be assembled into a singlecontiguous nucleotide sequence. As used herein, “substantially similar”refers to nucleic acid fragments wherein changes in one or morenucleotide bases results in substitution of one or more amino acids, butdo not affect the functional properties of the protein encoded by theDNA sequence.

“Substantially similar” also refers to nucleic acid fragments whereinchanges in one or more nucleotide bases does not affect the ability ofthe nucleic acid fragment to mediate alteration of gene expression byantisense or co-suppression technology. “Substantially similar” alsorefers to modifications of the nucleic acid fragments of the instantinvention such as deletion or insertion of one or more nucleotides thatdo not substantially affect the functional properties of the resultingtranscript vis-à-vis the ability to mediate alteration of geneexpression by antisense or co-suppression technology or alteration ofthe functional properties of the resulting protein molecule. It istherefore understood that the invention encompasses more than thespecific exemplary sequences.

For example, it is well known in the art that antisense suppression andco-suppression of gene expression may be accomplished using nucleic acidfragments representing less than the entire coding region of a gene, andby nucleic acid fragments that do not share 100% sequence identity withthe gene to be suppressed. Moreover, alterations in a gene which resultin the production of a chemically equivalent amino acid at a given site,but do not effect the functional properties of the encoded protein, arewell known in the art. Thus, a codon for the amino acid alanine, ahydrophobic amino acid, may be substituted by a codon encoding anotherless hydrophobic residue, such as glycine, or a more hydrophobicresidue, such as valine, leucine, or isoleucine. Similarly, changeswhich result in substitution of one negatively charged residue foranother, such as aspartic acid for glutamic acid, or one positivelycharged residue for another, such as lysine for arginine, can also beexpected to produce a functionally equivalent product. Nucleotidechanges which result in alteration of the N-terminal and C-terminalportions of the protein molecule would also not be expected to alter theactivity of the protein. Each of the proposed modifications is wellwithin the routine skill in the art, as is determination of retention ofbiological activity of the encoded products.

Moreover, substantially similar nucleic acid fragments may also becharacterized by their ability to hybridize, under stringent conditions(0.1×SSC, 0.1% SDS, 65° C.), with the nucleic acid fragments disclosedherein.

Substantially similar nucleic acid fragments of the instant inventionmay also be characterized by the percent similarity of the amino acidsequences that they encode to the amino acid sequences disclosed herein,as determined by algorithms commonly employed by those skilled in thisart. Preferred are those nucleic acid fragments whose nucleotidesequences encode amino acid sequences that are 90% similar to the aminoacid sequences reported herein. Most preferred are nucleic acidfragments that encode amino acid sequences that are 95% similar to theamino acid sequences reported herein. Sequence alignments and percentsimilarity calculations were performed using the Megalign program of theLASARGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).Multiple alignment of the sequences was performed using the Clustalmethod of alignment (Higgins, D. G. and Sharp, P. M. (1989) CABIOS.5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTHPENALTY=10), (hereafter Clustal algorithm). Default parameters forpairwise alignments using the Clustal method were KTUPLE 1, GAPPENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

A “substantial portion” of an amino acid or nucleotide sequencecomprises enough of the amino acid sequence of a polypeptide or thenucleotide sequence of a gene to afford putative identification of thatpolypeptide or gene, either by manual evaluation of the sequence by oneskilled in the art, or by computer-automated sequence comparison andidentification using algorithms such as BLAST (Basic Local AlignmentSearch Tool; Altschul, S. F., et al., (1993) J. Mol. Biol. 215:403-410.In general, a sequence of ten or more contiguous amino acids or thirtyor more nucleotides is necessary in order to putatively identify apolypeptide or nucleic acid sequence as homologous to a known protein orgene. Moreover, with respect to nucleotide sequences, gene specificoligonucleotide probes comprising 20-30 contiguous nucleotides may beused in sequence-dependent methods of gene identification (e.g.,Southern hybridization) and isolation (e.g., in situ hybridization ofbacterial colonies or bacteriophage plaques). In addition, shortoligonucleotides of 12-15 bases may be used as amplification primers inPCR in order to obtain a particular nucleic acid fragment comprising theprimers. Accordingly, a “substantial portion” of a nucleotide sequencecomprises enough of the sequence to afford specific identificationand/or isolation of a nucleic acid fragment comprising the sequence. Theinstant specification teaches partial or complete amino acid andnucleotide sequences encoding one or more particular plant proteins. Theskilled artisan, having the benefit of the sequences as reported herein,may now use all or a substantial portion of the disclosed sequences forpurposes known to those skilled in this art. Accordingly, the instantinvention comprises the complete sequences as reported in theaccompanying Sequence Listing, as well as substantial portions of thosesequences as defined above.

“Codon degeneracy” refers to divergence in the genetic code permittingvariation of the nucleotide sequence without effecting the amino acidsequence of an encoded polypeptide. Accordingly, the instant inventionrelates to any nucleic acid fragment that encodes all or a substantialportion of the amino acid sequence encoding the sucrose transportproteins as set forth in SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22 and 24. The skilled artisan is well aware of the “codon-bias”exhibited by a specific host cell in usage of nucleotide codons tospecify a given amino acid. Therefore, when synthesizing a gene forimproved expression in a host cell, it is desirable to design the genesuch that its frequency of codon usage approaches the frequency ofpreferred codon usage of the host cell.

“Synthetic genes” can be assembled from oligonucleotide building blocksthat are chemically synthesized using procedures known to those skilledin the art. These building blocks are ligated and annealed to form genesegments which are then enzymatically assembled to construct the entiregene. “Chemically synthesized”, as related to a sequence of DNA, meansthat the component nucleotides were assembled in vitro. Manual chemicalsynthesis of DNA may be accomplished using well established procedures,or automated chemical synthesis can be performed using one of a numberof commercially available machines. Accordingly, the genes can betailored for optimal gene expression based on optimization of nucleotidesequence to reflect the codon bias of the host cell. The skilled artisanappreciates the likelihood of successful gene expression if codon usageis biased towards those codons favored by the host. Determination ofpreferred codons can be based on a survey of genes derived from the hostcell where sequence information is available.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

“Coding sequence” refers to a DNA sequence that codes for a specificamino acid sequence. “Regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, and polyadenylationrecognition sequences.

“Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. The promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is aDNA sequence which can stimulate promoter activity and may be an innateelement of the promoter or a heterologous element inserted to enhancethe level or tissue-specificity of a promoter. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental conditions. Promoters whichcause a gene to be expressed in most cell types at most times arecommonly referred to as “constitutive promoters”. New promoters ofvarious types useful in plant cells are constantly being discovered;numerous examples may be found in the compilation by Okamuro andGoldberg, (1989) Biochemistry of Plants 15:1-82. It is furtherrecognized that since in most cases the exact boundaries of regulatorysequences have not been completely defined, DNA fragments of differentlengths may have identical promoter activity.

The “translation leader sequence” refers to a DNA sequence locatedbetween the promoter sequence of a gene and the coding sequence. Thetranslation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner, R. and Foster, G. D. (1995)Molecular Biotechnology 3:225).

The “3′ non-coding sequences” refer to DNA sequences located downstreamof a coding sequence and include polyadenylation recognition sequencesand other sequences encoding regulatory signals capable of affectingmRNA processing or gene expression. The polyadenylation signal isusually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht et al., (1989) PlantCell 1:671-680.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated into proteinby the cell. “cDNA” refers to a double-stranded DNA that iscomplementary to and derived from mRNA. “Sense” RNA refers to RNAtranscript that includes the mRNA and so can be translated into proteinby the cell. “Antisense RNA” refers to a RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target gene (U.S. Pat. No. 5,107,065,incorporated herein by reference). The complementarity of an antisenseRNA may be with any part of the specific gene transcript, i.e., at the5′ non-coding sequence, 3′ non-coding sequence, introns, or the codingsequence. “Functional RNA” refers to sense RNA, antisense RNA, ribozymeRNA, or other RNA that may not be translated but yet has an effect oncellular processes.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment of the invention. Expression may also refer totranslation of mRNA into a polypeptide. “Antisense inhibition” refers tothe production of antisense RNA transcripts capable of suppressing theexpression of the target protein. “Overexpression” refers to theproduction of a gene product in transgenic organisms that exceeds levelsof production in normal or non-transformed organisms. “Co-suppression”refers to the production of sense RNA transcripts capable of suppressingthe expression of identical or substantially similar foreign orendogenous genes (U.S. Pat. No. 5,231,020, incorporated herein byreference).

“Altered levels” refers to the production of gene product(s) intransgenic organisms in amounts or proportions that differ from that ofnormal or non-transformed organisms.

“Mature” protein refers to a post-translationally processed polypeptide;i.e., one from which any pre- or propeptides present in the primarytranslation product have been removed. “Precursor” protein refers to theprimary product of translation of mRNA; i.e., with pre- and propeptidesstill present. Pre- and propeptides may be but are not limited tointracellular localization signals.

A “chloroplast transit peptide” is an amino acid sequence which istranslated in conjunction with a protein and directs the protein to thechloroplast or other plastid types present in the cell in which theprotein is made. “Chloroplast transit sequence” refers to a nucleotidesequence that encodes a chloroplast transit peptide. A “signal peptide”is an amino acid sequence which is translated in conjunction with aprotein and directs the protein to the secretory system (Chrispeels, J.J., (1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If theprotein is to be directed to a vacuole, a vacuolar targeting signal(supra) can further be added, or if to the endoplasmic reticulum, anendoplasmic reticulum retention signal (supra) may be added. If theprotein is to be directed to the nucleus, any signal peptide presentshould be removed and instead a nuclear localization signal included(Raikhel (1992) Plant Phys. 100:1627-1632).

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms. Examples of methodsof plant transformation include Agrobacterium-mediated transformation(De Blaere et al. (1987) Meth. Enzymol. 143:277) andparticle-accelerated or “gene gun” transformation technology (Klein etal. (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050,incorporated herein by reference).

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook, J.,Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual;Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989(hereinafter “Maniatis”).

Nucleic acid fragments encoding at least a portion of several sucrosetransport proteins have been isolated and identified by comparison ofrandom plant cDNA sequences to public databases containing nucleotideand protein sequences using the BLAST algorithms well known to thoseskilled in the art. Table 1 lists the proteins that are describedherein, and the designation of the cDNA clones that comprise the nucleicacid fragments encoding these proteins.

TABLE 1 Sucrose Transport Proteins Enzyme Clone Plant SucroseTransporter cepe7.pk0015.d10 Corn cr1n.pk0095.c10 Corn cr1n.pk0075.f5Corn rlr2.pk0043.b1 Rice rls6.pk0076.e2 Rice sfl1.pk0001.g1 Soybeansfl1.pk0043.c7 Soybean sdp3c.pk012.c13 Soybean vs1n.pk0002.h3 Vernoniawle1n.pk0007.h8 Wheat wle1n.pk0103.c11 Wheat wlm24.pk0015.g11 Wheatwlmk1.pk0002.e11 Wheat

The nucleic acid fragments of the instant invention may be used toisolate cDNAs and genes encoding homologous proteins from the same orother plant species. Isolation of homologous genes usingsequence-dependent protocols is well known in the art. Examples ofsequence-dependent protocols include, but are not limited to, methods ofnucleic acid hybridization, and methods of DNA and RNA amplification asexemplified by various uses of nucleic acid amplification technologies(e.g., polymerase chain reaction, ligase chain reaction).

For example, genes encoding other sucrose transport proteins, either ascDNAs or genomic DNAs, could be isolated directly by using all or aportion of the instant nucleic acid fragments as DNA hybridizationprobes to screen libraries from any desired plant employing methodologywell known to those skilled in the art. Specific oligonucleotide probesbased upon the instant nucleic acid sequences can be designed andsynthesized by methods known in the art (Maniatis). Moreover, the entiresequences can be used directly to synthesize DNA probes by methods knownto the skilled artisan such as random primer DNA labeling, nicktranslation, or end-labeling techniques, or RNA probes using availablein vitro transcription systems. In addition, specific primers can bedesigned and used to amplify a part or all of the instant sequences. Theresulting amplification products can be labeled directly duringamplification reactions or labeled after amplification reactions, andused as probes to isolate full length cDNA or genomic fragments underconditions of appropriate stringency.

In addition, two short segments of the instant nucleic acid fragmentsmay be used in polymerase chain reaction protocols to amplify longernucleic acid fragments encoding homologous genes from DNA or RNA. Thepolymerase chain reaction may also be performed on a library of clonednucleic acid fragments wherein the sequence of one primer is derivedfrom the instant nucleic acid fragments, and the sequence of the otherprimer takes advantage of the presence of the polyadenylic acid tractsto the 3′ end of the mRNA precursor encoding plant genes. Alternatively,the second primer sequence may be based upon sequences derived from thecloning vector. For example, the skilled artisan can follow the RACEprotocol (Frohman et al., (1988) PNAS USA 85:8998) to generate cDNAs byusing PCR to amplify copies of the region between a single point in thetranscript and the 3′ or 5′ end. Primers oriented in the 3′ and 5′directions can be designed from the instant sequences. Usingcommercially available 3′ RACE or 5′ RACE systems (BRL), specific 3′ or5′ cDNA fragments can be isolated (Ohara et al., (1989) PNAS USA86:5673; Loh et al., (1989) Science 243:217). Products generated by the3′ and 5′ RACE procedures can be combined to generate full-length cDNAs(Frohman, M. A. and Martin, G. R., (1989) Techniques 1:165).

Availability of the instant nucleotide and deduced amino acid sequencesfacilitates immunological screening of cDNA expression libraries.Synthetic peptides representing portions of the instant amino acidsequences may be synthesized. These peptides can be used to immunizeanimals to produce polyclonal or monoclonal antibodies with specificityfor peptides or proteins comprising the amino acid sequences. Theseantibodies can be then be used to screen cDNA expression libraries toisolate full-length cDNA clones of interest (Lerner, R. A. (1984) Adv.Immunol. 36:1; Maniatis).

The nucleic acid fragments of the instant invention may be used tocreate transgenic plants in which the disclosed sucrose transportproteins are present at higher or lower levels than normal or in celltypes or developmental stages in which they are not normally found. Thiswould have the effect of altering the level of sucrose metabolism inthose cells.

Overexpression of the sucrose transport proteins of the instantinvention may be accomplished by first constructing a chimeric gene inwhich the coding region is operably linked to a promoter capable ofdirecting expression of a gene in the desired tissues at the desiredstage of development. For reasons of convenience, the chimeric gene maycomprise promoter sequences and translation leader sequences derivedfrom the same genes. 3′ Non-coding sequences encoding transcriptiontermination signals may also be provided. The instant chimeric gene mayalso comprise one or more introns in order to facilitate geneexpression.

Plasmid vectors comprising the instant chimeric gene can thenconstructed. The choice of plasmid vector is dependent upon the methodthat will be used to transform host plants. The skilled artisan is wellaware of the genetic elements that must be present on the plasmid vectorin order to successfully transform, select and propagate host cellscontaining the chimeric gene. The skilled artisan will also recognizethat different independent transformation events will result indifferent levels and patterns of expression (Jones et al., (1985) EMBOJ. 4:2411-2418; De Almeida et al., (1989) Mol. Gen. Genetics 218:78-86),and thus that multiple events must be screened in order to obtain linesdisplaying the desired expression level and pattern. Such screening maybe accomplished by Southern analysis of DNA, Northern analysis of mRNAexpression, Western analysis of protein expression, or phenotypicanalysis.

For some applications it may be useful to direct the instant sucrosetransport proteins to different cellular compartments, or to facilitateits secretion from the cell. It is thus envisioned that the chimericgene described above may be further supplemented by altering the codingsequence to encode a sucrose transport protein with appropriateintracellular targeting sequences such as transit sequences (Keegstra,K. (1989) Cell 56:247-253), signal sequences or sequences encodingendoplasmic reticulum localization (Chrispeels, J. J., (1991) Ann. Rev.Plant Phys. Plant Mol. Biol. 42:21-53), or nuclear localization signals(Raikhel, N. (1992) Plant Phys. 100:1627-1632) added and/or withtargeting sequences that are already present removed. While thereferences cited give examples of each of these, the list is notexhaustive and more targeting signals of utility may be discovered inthe future.

It may also be desirable to reduce or eliminate expression of genesencoding sucrose transport proteins in plants for some applications. Inorder to accomplish this, a chimeric gene designed for co-suppression ofthe instant sucrose transport proteins can be constructed by linking agene or gene fragment encoding a sucrose transport protein to plantpromoter sequences. Alternatively, a chimeric gene designed to expressantisense RNA for all or part of the instant nucleic acid fragment canbe constructed by linking the gene or gene fragment in reverseorientation to plant promoter sequences. Either the co-suppression orantisense chimeric genes could be introduced into plants viatransformation wherein expression of the corresponding endogenous genesare reduced or eliminated.

The instant sucrose transport proteins (or portions thereof) may beproduced in heterologous host cells, particularly in the cells ofmicrobial hosts, and can be used to prepare antibodies to the theseproteins by methods well known to those skilled in the art. Theantibodies are useful for detecting sucrose transport proteins in situin cells or in vitro in cell extracts. Preferred heterologous host cellsfor production of the instant sucrose transport proteins are microbialhosts. Microbial expression systems and expression vectors containingregulatory sequences that direct high level expression of foreignproteins are well known to those skilled in the art. Any of these couldbe used to construct a chimeric gene for production of the instantsucrose transport proteins. This chimeric gene could then be introducedinto appropriate microorganisms via transformation to provide high levelexpression of the encoded sucrose transport protein. An example of avector for high level expression of the instant sucrose transportproteins in a bacterial host is provided (Example 6).

All or a substantial portion of the nucleic acid fragments of theinstant invention may also be used as probes for genetically andphysically mapping the genes that they are a part of, and as markers fortraits linked to those genes. Such information may be useful in plantbreeding in order to develop lines with desired phenotypes. For example,the instant nucleic acid fragments may be used as restriction fragmentlength polymorphism (RFLP) markers. Southern blots (Maniatis) ofrestriction-digested plant genomic DNA may be probed with the nucleicacid fragments of the instant invention. The resulting banding patternsmay then be subjected to genetic analyses using computer programs suchas MapMaker (Lander et at., (1987) Genomics 1:174-181) in order toconstruct a genetic map. In addition, the nucleic acid fragments of theinstant invention may be used to probe Southern blots containingrestriction endonuclease-treated genomic DNAs of a set of individualsrepresenting parent and progeny of a defined genetic cross. Segregationof the DNA polymorphisms is noted and used to calculate the position ofthe instant nucleic acid sequence in the genetic map previously obtainedusing this population (Botstein, D. et al., (1980) Am. J. Hum. Genet.32:314-331).

The production and use of plant gene-derived probes for use in geneticmapping is described in R. Bernatzky, R. and Tanksley, S. D. (1986)Plant Mol. Biol. Reporter 4(1):37-41. Numerous publications describegenetic mapping of specific cDNA clones using the methodology outlinedabove or variations thereof. For example, F2 intercross populations,backcross populations, randomly mated populations, near isogenic lines,and other sets of individuals may be used for mapping. Suchmethodologies are well known to those skilled in the art.

Nucleic acid probes derived from the instant nucleic acid sequences mayalso be used for physical mapping (i.e., placement of sequences onphysical maps; see Hoheisel, J. D., et al., In: Nonmammalian GenomicAnalysis: A Practical Guide, Academic press 1996, pp. 319-346, andreferences cited therein).

In another embodiment, nucleic acid probes derived from the instantnucleic acid sequences may be used in direct fluorescence in situhybridization (FISH) mapping (Trask, B. J. (1991) Trends Genet.7:149-154). Although current methods of FISH mapping favor use of largeclones (several to several hundred KB; see Laan, M. et al. (1995) GenomeResearch 5:13-20), improvements in sensitivity may allow performance ofFISH mapping using shorter probes.

A variety of nucleic acid amplification-based methods of genetic andphysical mapping may be carried out using the instant nucleic acidsequences. Examples include allele-specific amplification (Kazazian, H.H. (1989) J. Lab. Clin. Med. 114(2):95-96), polymorphism ofPCR-amplified fragments (CAPS; Sheffield, V. C. et al. (1993) Genomics16:325-332), allele-specific ligation (Landegren, U. et al. (1988)Science 241:1077-1080), nucleotide extension reactions (Sokolov, B. P.(1990) Nucleic Acid Res. 18:3671), Radiation Hybrid Mapping (Walter, M.A. et al. (1997) Nature Genetics 7:22-28) and Happy Mapping (Dear, P. H.and Cook, P. R. (1989) Nucleic Acid Res. 17:6795-6807). For thesemethods, the sequence of a nucleic acid fragment is used to design andproduce primer pairs for use in the amplification reaction or in primerextension reactions. The design of such primers is well known to thoseskilled in the art. In methods employing PCR-based genetic mapping, itmay be necessary to identify DNA sequence differences between theparents of the mapping cross in the region corresponding to the instantnucleic acid sequence. This, however, is generally not necessary formapping methods.

Loss of function mutant phenotypes may be identified for the instantcDNA clones either by targeted gene disruption protocols or byidentifying specific mutants for these genes contained in a maizepopulation carrying mutations in all possible genes (Ballinger andBenzer, (1989) Proc. Natl. Acad. Sci USA 86:9402; Koes et al., (1995)Proc. Natl. Acad. Sci USA 92:8149; Bensen et al., (1995) Plant Cell7:75). The latter approach may be accomplished in two ways. First, shortsegments of the instant nucleic acid fragments may be used in polymerasechain reaction protocols in conjunction with a mutation tag sequenceprimer on DNAs prepared from a population of plants in which Mutatortransposons or some other mutation-causing DNA element has beenintroduced (see Bensen, supra). The amplification of a specific DNAfragment with these primers indicates the insertion of the mutation tagelement in or near the plant gene encoding the sucrose transportprotein. Alternatively, the instant nucleic acid fragment may be used asa hybridization probe against PCR amplification products generated fromthe mutation population using the mutation tag sequence primer inconjunction with an arbitrary genomic site primer, such as that for arestriction enzyme site-anchored synthetic adaptor. With either method,a plant containing a mutation in the endogenous gene encoding a sucrosetransport protein can be identified and obtained. This mutant plant canthen be used to determine or confirm the natural function of the sucrosetransport protein gene product.

EXAMPLES

The present invention is further defined in the following Examples, inwhich all parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these Examples,while indicating preferred embodiments of the invention, are given byway of illustration only. From the above discussion and these Examples,one skilled in the art can ascertain the essential characteristics ofthis invention, and without departing from the spirit and scope thereof,can make various changes and modifications of the invention to adapt itto various usages and conditions.

Example 1 Composition of cDNA Libraries; Isolation and Sequencing ofcDNA Clones

cDNA libraries representing mRNAs from various corn, rice, soybean,Vernonia and wheat tissues were prepared. The characteristics of thelibraries are described below.

TABLE 2 cDNA Libraries from Corn Rice, Soybean Vernonia and WheatLibrary Tissue Clone cepe7 Corn epicotyl from 7 day old etiolatedcepe7.pk0015.d10 seedling cr1n Corn root from 7 day seedling grown incr1n.pk0075.f5 light* cr1n.pk0095.c10 rlr2 Rice leaf 15 days aftergermination rlr2.pk0043.b1 2 hours after infection of strain Magnaporthegrisea 4360-R-62 (AVR2-YAMO) rls6 Rice leaf 15 days after germinationrls6.pk0076.e2 6 hours after infection of strain Magnaporthe grisea4360-R-62 (AVR2-YAMO) sdp3c Soybean developing pods 8-9 mmsdp3c.pk012.c13 sfl1 Soybean immature flower sfl1.pk0001.g1sfl1.pk0043.c7 vs1 Vernonia developing seed vs1n.pk0002.h3 wle1n Wheatleaf 7 day old etiolated seedling wle1n.pk0007.h8 light grown*wle1n.pk0103.c11 wlm24 Wheat seedling 24 hours after inoculationwlm24.pk0015.g11 with Erysiphe graminis wlmk1 Wheat seedlings 1 hourafter inoculation wlmk1.pk0002.e11 with Erysiphe graminis and treatmentwith fungicide** *These libraries were normalized essentially asdescribed in U.S. Pat. No. 5,482,845 **Application of6-iodo-2-propoxy-3-propyl-4(3H)-quinazolinone; synthesis and methods ofusing this compound are described in U.S. Ser. No. 08/545,827,incorporated herein by reference.

cDNA libraries were prepared in Uni-ZAP™ XR vectors according to themanufacturer's protocol (Stratagene Cloning Systems, La Jolla, Calif.).Conversion of the Uni-ZAP™ XR libraries into plasmid libraries wasaccomplished according to the protocol provided by Stratagene. Uponconversion, cDNA inserts were contained in the plasmid vectorpBluescript. cDNA inserts from randomly picked bacterial coloniescontaining recombinant pBluescript plasmids were amplified viapolymerase chain reaction using primers specific for vector sequencesflanking the inserted cDNA sequences or plasmid DNA was prepared fromcultured bacterial cells. Amplified insert DNAs or plasmid DNAs weresequenced in dye-primer sequencing reactions to generate partial cDNAsequences (expressed sequence tags or “ESTs”; see Adams, M. D. et al.,(1991) Science 252:1651). The resulting ESTs were analyzed using aPerkin Elmer Model 377 fluorescent sequencer.

Example 2 Identification of cDNA Clones

ESTs encoding sucrose transport proteins were identified by conductingBLAST (Basic Local Alignment Search Tool; Altschul, S. F., et al.,(1993) J. Mol. Biol. 215:403-410 searches for similarity to sequencescontained in the BLAST “nr” database (comprising all non-redundantGenBank CDS translations, sequences derived from the 3-dimensionalstructure Brookhaven Protein Data Bank, the last major release of theSWISS-PROT protein sequence database, EMBL, and DDBJ databases). ThecDNA sequences obtained in Example 1 were analyzed for similarity to allpublicly available DNA sequences contained in the “nr” database usingthe BLASTN algorithm provided by the National Center for BiotechnologyInformation (NCBI). The DNA sequences were translated in all readingframes and compared for similarity to all publicly available proteinsequences contained in the “nr” database using the BLASTX algorithm(Gish, W. and States, D. J. (1993) Nature Genetics 3:266-272 andAltschul, Stephen F., et al. (1997) Nucleic Acids Res. 25:3389-3402)provided by the NCBI. For convenience, the P-value (probability) ofobserving a match of a cDNA sequence to a sequence contained in thesearched databases merely by chance as calculated by BLAST are reportedherein as “pLog” values, which represent the negative of the logarithmof the reported P-value. Accordingly, the greater the pLog value, thegreater the likelihood that the cDNA sequence and the BLAST “hit”represent homologous proteins.

Example 3 Characterization of cDNA Clones Encoding Sucrose TransporterProteins

The BLASTX search using the EST sequences from clones cepe7.pk0015.d10,cr1n.pk0095.c10, cr1n.pk0075.f5, r1s6.pk0076.e2, w1e1n.pk0007.h8,w1e1n.pk0007.h8, w1e1n.pk0103.c11, w1m24.pk0015.g11 and w1mk1.pk0002.e11revealed similarity of the proteins encoded by the cDNAs to a sucrosetransporter from Oryza sativa (NCBI Identifier No. gi 2723471). TheBLASTX search using the EST sequence from clone r1r2.pk0043.b1 revealedsimilarity of the protein encoded by the cDNA to a sucrose transporterfrom Daucus carota (NCBI Identifier No. gi 2969887). The BLASTX searchusing the EST sequence from clone sfl1.pk0001.g1 revealed similarity ofthe protein encoded by the cDNA to a sucrose transporter from Vicia faba(NCBI Identifier No. gi 1935019). The BLASTX search using the ESTsequences from clones sfl1.pk0043.c7, sdp3c.pk012.c13 and vs1n.pk0002.h3revealed similarity of the proteins encoded by the cDNAs to a sucrosetransporter from Ricinus communis (NCBI Identifier No. gi 542020).

In the process of comparing the ESTs it was found that soybean clonessfl1.pk0043.c7 and sdp3c.pk012.c13 had overlapping regions of homology.Using this homology it was possible to align the ESTs and assemble acontig encoding a unique soybean sucrose transport protein.

The BLAST results for each of these ESTs and the soybean contig areshown in Table 3:

TABLE 3 BLAST Results for Clones Encoding Polypeptides Homologous toDaucus carota, Oryza sativa, Ricinus communis and Vicia faba SucroseTransport Proteins Clone BLAST pLog Score cepe7.pk0005.d10 >250.00cr1n.pk0095.c10 >250.00 cr1n.pk0075.f5 31.10 rlr2.pk0043.b1 148.00rls6.pk0076.e2 >250.00 sfl1.pk0001.g1 >250.00 Contig composed of: 142.00sfl1.pk0043.c7 sdp3c.pk012.c13 vs1n.pk0002.h3 59.30 wle1n.pk0007.h8110.00 wle1n.pk0103.c11 >250.00 wlm24.pk0015.g11 >250.00wlmk1.pk0002.e11 177.00

The sequence of a portion of the cDNA insert from clone cepe7.pk0015.d10is shown in SEQ ID NO:1; the deduced amino acid sequence of this cDNA,which represents 100% of the protein, is shown in SEQ ID NO:2. Acalculation of the percent similarity of the amino acid sequence setforth in SEQ ID NO:2 and the Oryza sativa sequence (using the Clustalalgorithm) revealed that the protein encoded by SEQ ID NO:2 is 82%similar to the Oryza sativa protein.

The sequence of a portion of the cDNA insert from clone cr1n.pk0075.f5is shown in SEQ ID NO:3; the deduced amino acid sequence of this cDNA,which represents 93% of the protein, is shown in SEQ ID NO:4. Acalculation of the percent similarity of the amino acid sequence setforth in SEQ ID NO:4 and the Oryza sativa sequence (using the Clustalalgorithm) revealed that the protein encoded by SEQ ID NO:4 is 50%similar to the Oryza sativa protein.

The sequence of a portion of the cDNA insert from clone cr1n.pk0095.c10is shown in SEQ ID NO:5; the deduced amino acid sequence of this cDNA,which represents 20% of the protein (C-terminal region), is shown in SEQID NO:6. A calculation of the percent similarity of the amino acidsequence set forth in SEQ ID NO:6 and the Oryza sativa sequence (usingthe Clustal algorithm) revealed that the protein encoded by SEQ ID NO:6is 86% similar to the Oryza sativa protein.

The sequence of a portion of the cDNA insert from clone r1r2.pk0043.b1is shown in SEQ ID NO:7; the deduced amino acid sequence of this cDNA,which represents 79% of the protein (C-terminal region), is shown in SEQID NO:8. A calculation of the percent similarity of the amino acidsequence set forth in SEQ ID NO:8 and the Daucus carota sequence (usingthe Clustal algorithm) revealed that the protein encoded by SEQ ID NO:8is 60% similar to the Daucus carota protein.

The sequence of a portion of the cDNA insert from clone r1s6.pk0076.e2is shown in SEQ ID NO:9; the deduced amino acid sequence of this cDNA,which represents 100% of the protein, is shown in SEQ ID NO:10. Acalculation of the percent similarity of the amino acid sequence setforth in SEQ ID NO:10 and the Oryza sativa sequence (using the Clustalalgorithm) revealed that the protein encoded by SEQ ID NO:10 is 55%similar to the Oryza sativa protein. Due to a percent similarity of only55% with a known rice sucrose transport protein clone r1s6.pk0076.e2appears to represent a second rice sucrose transport protein.

The sequence of a portion of the cDNA insert from clone sfl1.pk0001.g1is shown in SEQ ID NO: 11; the deduced amino acid sequence of this cDNA,which represents 100% of the protein, is shown in SEQ ID NO:12. Acalculation of the percent similarity of the amino acid sequence setforth in SEQ ID NO:12 and the Vicia faba sequence (using the Clustalalgorithm) revealed that the protein encoded by SEQ ID NO:12 is 67%similar to the Vicia faba protein.

The sequence of a portion of the contig composed of clonessfl1.pk0043.c7 and sdp3c.pk012.c13 is shown in SEQ ID NO:13; the deducedamino acid sequence of this contig, which represents 62% of the protein(N-terminal region), is shown in SEQ ID NO:14. A calculation of thepercent similarity of the amino acid sequence set forth in SEQ ID NO:14and the Ricinus communis sequence (using the Clustal algorithm) revealedthat the protein encoded by SEQ ID NO:14 is 66% similar to the Ricinuscommunes protein.

The sequence of a portion of the cDNA insert from clone vs1n.pk0002.h3is shown in SEQ ID NO:15; the deduced amino acid sequence of this cDNA,which represents 31% of the protein (C-terminal region), is shown in SEQID NO:16. A calculation of the percent similarity of the amino acidsequence set forth in SEQ ID NO:16 and the Ricinus communes sequence(using the Clustal algorithm) revealed that the protein encoded by SEQID NO:16 is 66% similar to the Ricinus communes protein.

The sequence of a portion of the cDNA insert from clone w1e1n.pk0007.h8is shown in SEQ ID NO:17; the deduced amino acid sequence of this cDNA,which represents 43% of the protein (C-terminal region), is shown in SEQID NO:18. A calculation of the percent similarity of the amino acidsequence set forth in SEQ ID NO:18 and the Oryza sativa sequence (usingthe Clustal algorithm) revealed that the protein encoded by SEQ ID NO:18is 80% similar to the Oryza sativa protein.

The sequence of a portion of the cDNA insert from clone w1e1n.pk0103.c11is shown in SEQ ID NO:19; the deduced amino acid sequence of this cDNA,which represents 100% of the protein, is shown in SEQ ID NO:20. Acalculation of the percent similarity of the amino acid sequence setforth in SEQ ID NO:20 and the Oryza sativa sequence (using the Clustalalgorithm) revealed that the protein encoded by SEQ ID NO:20 is 80%similar to the Oryza sativa protein.

The sequence of a portion of the cDNA insert from clone w1m24.pk0015.g11is shown in SEQ ID NO:21; the deduced amino acid sequence of this cDNA,which represents 100% of the protein, is shown in SEQ ID NO:22. Acalculation of the percent similarity of the amino acid sequence setforth in SEQ ID NO:22 and the Oryza sativa sequence (using the Clustalalgorithm) revealed that the protein encoded by SEQ ID NO:22 is 80%similar to the Oryza sativa protein.

The sequence of a portion of the cDNA insert from clone w1mk1.pk0002.e11is shown in SEQ ID NO:23; the deduced amino acid sequence of this cDNA,which represents 97% of the protein, is shown in SEQ ID NO:24. Acalculation of the percent similarity of the amino acid sequence setforth in SEQ ID NO:24 and the Oryza sativa sequence (using the Clustalalgorithm) revealed that the protein encoded by SEQ ID NO:24 is 54%similar to the Oryza sativa protein.

The percent similarity between each of the corn, rice, soybean, Vernoniaand wheat amino acid sequence was calculated to range from 12 to 98%using the Clustal algorithm. FIG. 1A-FIG. 1F presents an alignment ofthe amino acid sequences set forth in SEQ ID NOs:2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22 and 24 and the Daucus carota, Oryza sativa, Ricinuscommunis and Vicia faba sucrose transport protein amino acid sequences.

BLAST scores and probabilities indicate that the instant nucleic acidfragments encode entire or portions of proteins. These sequencesrepresent the first corn, soybean and wheat, amino acid sequences and anew rice sequence encoding sucrose transport proteins.

Example 4 Expression of Chimeric Genes in Monocot Cells

A chimeric gene comprising a cDNA encoding a sucrose transport proteinin sense orientation with respect to the maize 27 kD zein promoter thatis located 5′ to the cDNA fragment, and the 10 kD zein 3′ end that islocated 3′ to the cDNA fragment, can be constructed. The cDNA fragmentof this gene may be generated by polymerase chain reaction (PCR) of thecDNA clone using appropriate oligonucleotide primers. Cloning sites(NcoI or SmaI) can be incorporated into the oligonucleotides to provideproper orientation of the DNA fragment when inserted into the digestedvector pML103 as described below. Amplification is then performed in astandard PCR. The amplified DNA is then digested with restrictionenzymes NcoI and SmaI and fractionated on an agarose gel. Theappropriate band can be isolated from the gel and combined with a 4.9 kbNcoI-SmaI fragment of the plasmid pML103. Plasmid pML103 has beendeposited under the terms of the Budapest Treaty at ATCC (American TypeCulture Collection, 10801 University Blvd., Manassas, Va. 20110-2209),and bears accession number ATCC 97366. The DNA segment from pML103contains a 1.05 kb SalI-NcoI promoter fragment of the maize 27 kD zeingene and a 0.96 kb SmaI-SalI fragment from the 3′ end of the maize 10 kDzein gene in the vector pGem9Zf(+) (Promega). Vector and insert DNA canbe ligated at 15° C. overnight, essentially as described (Maniatis). Theligated DNA may then be used to transform E. coli XL1-Blue (EpicurianColi XL-1 Blue™; Stratagene). Bacterial transformants can be screened byrestriction enzyme digestion of plasmid DNA and limited nucleotidesequence analysis using the dideoxy chain termination method (Sequenase™DNA Sequencing Kit; U.S. Biochemical). The resulting plasmid constructwould comprise a chimeric gene encoding, in the 5′ to 3′ direction, themaize 27 kD zein promoter, a cDNA fragment encoding a sucrose transportprotein, and the 10 kD zein 3′ region.

The chimeric gene described above can then be introduced into corn cellsby the following procedure. Immature corn embryos can be dissected fromdeveloping caryopses derived from crosses of the inbred corn lines H99and LH132. The embryos are isolated 10 to 11 days after pollination whenthey are 1.0 to 1.5 mm long. The embryos are then placed with theaxis-side facing down and in contact with agarose-solidified N6 medium(Chu et al., (1975) Sci. Sin. Peking 18:659-668). The embryos are keptin the dark at 27° C. Friable embryogenic callus consisting ofundifferentiated masses of cells with somatic proembryoids and embryoidsborne on suspensor structures proliferates from the scutellum of theseimmature embryos. The embryogenic callus isolated from the primaryexplant can be cultured on N6 medium and sub-cultured on this mediumevery 2 to 3 weeks.

The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag,Frankfurt, Germany) may be used in transformation experiments in orderto provide for a selectable marker. This plasmid contains the Pat gene(see European Patent Publication 0 242 236) which encodesphosphinothricin acetyl transferase (PAT). The enzyme PAT confersresistance to herbicidal glutamine synthetase inhibitors such asphosphinothricin. The pat gene in p35S/Ac is under the control of the35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature313:810-812) and the 3′ region of the nopaline synthase gene from theT-DNA of the Ti plasmid of Agrobacterium tumefaciens.

The particle bombardment method (Klein et al., (1987) Nature 327:70-73)may be used to transfer genes to the callus culture cells. According tothis method, gold particles (1 μm in diameter) are coated with DNA usingthe following technique. Ten μg of plasmid DNAs are added to 50 μL of asuspension of gold particles (60 mg per mL). Calcium chloride (50 μL ofa 2.5 M solution) and spermidine free base (20 μL of a 1.0 M solution)are added to the particles. The suspension is vortexed during theaddition of these solutions. After 10 minutes, the tubes are brieflycentrifuged (5 sec at 15,000 rpm) and the supernatant removed. Theparticles are resuspended in 200 μL of absolute ethanol, centrifugedagain and the supernatant removed. The ethanol rinse is performed againand the particles resuspended in a final volume of 30 μL of ethanol. Analiquot (5 μL) of the DNA-coated gold particles can be placed in thecenter of a Kapton™ flying disc (Bio-Rad Labs). The particles are thenaccelerated into the corn tissue with a Biolistic™ PDS-1000/He (Bio-RadInstruments, Hercules Calif.), using a helium pressure of 1000 psi, agap distance of 0.5 cm and a flying distance of 1.0 cm.

For bombardment, the embryogenic tissue is placed on filter paper overagarose-solidified N6 medium. The tissue is arranged as a thin lawn andcovered a circular area of about 5 cm in diameter. The petri dishcontaining the tissue can be placed in the chamber of the PDS-1000/Heapproximately 8 cm from the stopping screen. The air in the chamber isthen evacuated to a vacuum of 28 inches of Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1000 psi.

Seven days after bombardment the tissue can be transferred to N6 mediumthat contains gluphosinate (2 mg per liter) and lacks casein or proline.The tissue continues to grow slowly on this medium. After an additional2 weeks the tissue can be transferred to fresh N6 medium containinggluphosinate. After 6 weeks, areas of about 1 cm in diameter of activelygrowing callus can be identified on some of the plates containing theglufosinate-supplemented medium. These calli may continue to grow whensub-cultured on the selective medium.

Plants can be regenerated from the transgenic callus by firsttransferring clusters of tissue to N6 medium supplemented with 0.2 mgper liter of 2,4-D. After two weeks the tissue can be transferred toregeneration medium (Fromm et al., (1990) Bio/Technology 8:833-839).

Example 5 Expression of Chimeric Genes in Dicot Cells

A seed-specific expression cassette composed of the promoter andtranscription terminator from the gene encoding the β subunit of theseed storage protein phaseolin from the bean Phaseolus vulgaris (Doyleet al. (1986) J. Biol. Chem. 261:9228-9238) can be used for expressionof the instant sucrose transport proteins in transformed soybean. Thephaseolin cassette includes about 500 nucleotides upstream (5′) from thetranslation initiation codon and about 1650 nucleotides downstream (3′)from the translation stop codon of phaseolin. Between the 5′ and 3′regions are the unique restriction endonuclease sites Nco I (whichincludes the ATG translation initiation codon), Sma I, Kpn I and Xba I.The entire cassette is flanked by Hind III sites.

The cDNA fragment of this gene may be generated by polymerase chainreaction (PCR) of the cDNA clone using appropriate oligonucleotideprimers. Cloning sites can be incorporated into the oligonucleotides toprovide proper orientation of the DNA fragment when inserted into theexpression vector. Amplification is then performed as described above,and the isolated fragment is inserted into a pUC18 vector carrying theseed expression cassette.

Soybean embroys may then be transformed with the expression vectorcomprising a sequence encoding a sucrose transport protein. To inducesomatic embryos, cotyledons, 3-5 mm in length dissected from surfacesterilized, immature seeds of the soybean cultivar A2872, can becultured in the light or dark at 26° C. on an appropriate agar mediumfor 6-10 weeks. Somatic embryos which produce secondary embryos are thenexcised and placed into a suitable liquid medium. After repeatedselection for clusters of somatic embryos which multiplied as early,globular staged embryos, the suspensions are maintained as describedbelow.

Soybean embryogenic suspension cultures can maintained in 35 mL liquidmedia on a rotary shaker, 150 rpm, at 26° C. with florescent lights on a16:8 hour day/night schedule. Cultures are subcultured every two weeksby inoculating approximately 35 mg of tissue into 35 mL of liquidmedium.

Soybean embryogenic suspension cultures may then be transformed by themethod of particle gun bombardment (Kline et al. (1987) Nature (London)327:70, U.S. Pat. No. 4,945,050). A DuPont Biolistic™ PDS1000/HEinstrument (helium retrofit) can be used for these transformations.

A selectable marker gene which can be used to facilitate soybeantransformation is a chimeric gene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz et al. (1983) Gene 25:179-188) and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The seed expression cassette comprising the phaseolin 5′region, the fragment encoding the sucrose transport protein and thephaseolin 3′ region can be isolated as a restriction fragment. Thisfragment can then be inserted into a unique restriction site of thevector carrying the marker gene.

To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (inorder): 5 μL DNA (1 μg/μL), 20 μl spermidine (0.1 M), and 50 μL CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μL 70% ethanol andresuspended in 40 μL of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five μL of theDNA-coated gold particles are then loaded on each macro carrier disk.

Approximately 300400 mg of a two-week-old suspension culture is placedin an empty 60×15 mm petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5-10 plates of tissue are normally bombarded. Membrane rupture pressureis set at 1100 psi and the chamber is evacuated to a vacuum of 28 inchesmercury. The tissue is placed approximately 3.5 inches away from theretaining screen and bombarded three times. Following bombardment, thetissue can be divided in half and placed back into liquid and culturedas described above.

Five to seven days post bombardment, the liquid media may be exchangedwith fresh media, and eleven to twelve days post bombardment with freshmedia containing 50 mg/mL hygromycin. This selective media can berefreshed weekly. Seven to eight weeks post bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

Example 6 Expression of Chimeric Genes in Microbial Cells

The cDNAs encoding the instant sucrose transport proteins can beinserted into the T7 E. coli expression vector pBT430. This vector is aderivative of pET-3a (Rosenberg et al. (1987) Gene 56:125-135) whichemploys the bacteriophage T7 RNA polymerase/T7 promoter system. PlasmidpBT430 was constructed by first destroying the EcoR I and Hind III sitesin pET-3a at their original positions. An oligonucleotide adaptorcontaining EcoR I and Hind III sites was inserted at the BamH I site ofpET-3a. This created pET-3aM with additional unique cloning sites forinsertion of genes into the expression vector. Then, the Nde I site atthe position of translation initiation was converted to an Nco I siteusing oligonucleotide-directed mutagenesis. The DNA sequence of pET-3aMin this region, 5′-CATATGG, was converted to 5′-CCCATGG in pBT430.

Plasmid DNA containing a cDNA may be appropriately digested to release anucleic acid fragment encoding the protein. This fragment may then bepurified on a 1% NuSieve GTG™ low melting agarose gel (FMC). Buffer andagarose contain 10 μg/ml ethidium bromide for visualization of the DNAfragment. The fragment can then be purified from the agarose gel bydigestion with GELase™ (Epicentre Technologies) according to themanufacturer's instructions, ethanol precipitated, dried and resuspendedin 20 μL of water. Appropriate oligonucleotide adapters may be ligatedto the fragment using T4 DNA ligase (New England Biolabs, Beverly,Mass.). The fragment containing the ligated adapters can be purifiedfrom the excess adapters using low melting agarose as described above.The vector pBT430 is digested, dephosphorylated with alkalinephosphatase (NEB) and deproteinized with phenol/chloroform as describedabove. The prepared vector pBT430 and fragment can then be ligated at16° C. for 15 hours followed by transformation into DH5 electrocompetentcells (GIBCO BRL). Transformants can be selected on agar platescontaining LB media and 100 μg/mL ampicillin. Transformants containingthe gene encoding the sucrose transport protein are then screened forthe correct orientation with respect to the T7 promoter by restrictionenzyme analysis.

For high level expression, a plasmid clone with the cDNA insert in thecorrect orientation relative to the T7 promoter can be transformed intoE. coli strain BL21(DE3) (Studier et al. (1986) J. Mol. Biol.189:113-130). Cultures are grown in LB medium containing ampicillin (100mg/L) at 25° C. At an optical density at 600 nm of approximately 1, IPTG(isopropylthio-β-galactoside, the inducer) can be added to a finalconcentration of 0.4 mM and incubation can be continued for 3 h at 25°.Cells are then harvested by centrifugation and re-suspended in 50 μL of50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTT and 0.2 mM phenylmethylsulfonyl fluoride. A small amount of 1 mm glass beads can be addedand the mixture sonicated 3 times for about 5 seconds each time with amicroprobe sonicator. The mixture is centrifuged and the proteinconcentration of the supernatant determined. One μg of protein from thesoluble fraction of the culture can be separated by SDS-polyacrylamidegel electrophoresis. Gels can be observed for protein bands migrating atthe expected molecular weight.

1. An isolated polynucleotide comprising: (a) a nucleotide sequenceencoding a polypeptide having sucrose transport activity, wherein thepolypeptide has an amino acid sequence of at least 95% sequenceidentity, based on the Clustal method of alignment, when compared to SEQID NO:20, or; (b) a complement of the nucleotide sequence of (a),wherein the complement and the nucleotide sequence consist of the samenumber of nucleotides and are 100% complementary.
 2. The polynucleotideof claim 1 wherein the nucleotide sequence comprises SEQ ID NO:19.
 3. Arecombinant DNA construct comprising the polynucleotide of claim 1operably linked to at least one regulatory sequence.
 4. A transformedhost cell comprising the recombinant DNA construct of claim
 3. 5. Amethod of altering the level of expression of a sucrose transportprotein in a host cell comprising: (a) transforming a host cell with therecombinant DNA construct of claim 3; and (b) growing the transformedhost cell produced in step (a) under conditions that are suitable forexpression of the recombinant DNA construct wherein expression of therecombinant DNA construct results in production of altered levels of asucrose transport protein in the transformed host cell.
 6. A plantcomprising the recombinant DNA construct of claim 3.